Apoptosis is a kind of ordered cell death, which depends on the implementation of a cascade of molecular events that culminate in the total disintegration of the cell. In the late stage of apoptosis, and as a result of cell fragmentation, small membranous vesicles called apoptotic bodies are generated.
Under physiological conditions, apoptosis is involved in morphogenesis and in tissue remodeling during the development, and the regulation of the immune system and homeostasis. However, this process also plays a key role in the cell and tissue death that occurs in various pathologies, such as vascular diseases, neurodegenerative diseases and oncological diseases. The evaluation of the apoptotic processes, namely assessing the degree of apoptosis or apoptotic index in these diseases would be useful in daily clinical practice, since it would allow an earlier and more accurate prognosis, more effective monitoring of the evolution of the disease, and the selection of the most appropriate treatment for each patient.
However, the in vivo analysis of apoptosis that occurs in tissue has a clear limitation as it is currently necessary to obtain tissue samples of patients by interventionist methods, biopsy or surgery. Therefore, there is an urgent medical need for non-invasive procedures that allow the study of apoptosis, preferably in a quick, simple and quantitative manner.
In the state of the art, a large number of studies that focus on determining the death of a tissue, particularly through the collection and analysis of solid samples taken by non-invasive methods such as biopsies (tissue samples) can be found. The degree of apoptosis is determined by various techniques known by the person skilled in the art, such as detection of DNA fragmentation (TUNEL technique, for example) and detecting the phosphatidylserine present on the outside of the plasma membrane of apoptotic cells with Annexin V. However, in these methods the patients' samples are obtained by invasive methods. Moreover, in most cases primary cultures of the biopsies are taken and subsequently apoptosis is induced in order to isolate apoptotic bodies (WO 99/58645 A1), that is, the apoptotic bodies are isolated in non-physiological conditions (artificial induction of apoptosis).
Surprisingly, the authors of the present invention have developed a method for isolating apoptotic bodies through centrifugation of samples of body fluids. That is, these apoptotic bodies are isolated in physiological conditions, without previous artificial induction of apoptosis. Moreover, with this method up to more than 90% of all apoptotic bodies from the sample of body fluid can be isolated and, besides, the apoptotic bodies maintain their integrity and are therefore detectable and measurable. In this sense, WO 03/076589 A2 describes methods for detecting and analysing apoptotic bodies from body fluids. Among the various methods described, there is one in which apoptotic bodies from the cell fraction are separated by centrifugation. In a preferred embodiment, the biological sample is blood and it is centrifuged at more than 500 g, preferably between 800 g and 1,200 g, so that the apoptotic bodies remain in the plasma fraction. If it is desirable to separate or isolate these apoptotic bodies from the plasma, another high speed centrifugation is performed (speed not defined, for the person skilled in the art of the present invention, a high speed corresponds to a speed higher than 60,000 g), after which the apoptotic bodies remain in the sediment. However, unlike what is achieved with the method of the present invention, with the conditions described in WO 03/076589 it is not possible to isolate up to 90% of all the apoptotic bodies present in the body fluids, nor maintain their integrity. First, this is due to the fact that a centrifugation speed higher than 500 g, aimed at obtaining plasma or serum, leads to the sedimentation of all cellular blood components, among which are platelets. These cells easily form aggregates, especially in the presence of other kind of blood cells such as erythrocytes and leukocytes. If this occurs, the apoptotic bodies would be retained in the cell aggregates and centrifugation at a speed higher than 500 g would lead to their sedimentation with the cell fraction, and this would mean losing a large number of them. Furthermore, the high speed centrifugation of plasma or serum obtained from the first centrifugation could result in rupture of the apoptotic bodies and in their contamination with other smaller membrane vesicles. Due to these drawbacks, the apoptotic bodies isolated by the method described in WO 03/076589 A2 have little or no utility for prognosis and/or monitoring of the treatment for subjects with diseases associated with apoptosis, as the number of isolated apoptotic bodies would be much lower than the real number in the body fluid and the apoptotic bodies could be disintegrated and/or contaminated by other microvesicles, resulting in an erroneous quantification of apoptotic bodies.
Surprisingly, the authors of the present invention have developed a method for isolating apoptotic bodies that maintain their integrity and can therefore be quantified. Furthermore, the method of the invention minimizes to the maximum the loss of apoptotic bodies during isolation and achieves the isolation of more than 90% of all apoptotic bodies present in the body fluid sample. In this way, the authors of the present invention have developed a very useful tool for the study of apoptosis. Thus, the present invention also relates to a prognosis method based on the quantification of isolated apoptotic bodies by the method of the invention and to the use of such apoptotic bodies as a prognostic factor and as a marker of the efficacy of a therapeutic treatment. Finally, another important advantage of the integrity of apoptotic bodies isolated by the method of the present invention is its use in order to identify the cell type of the cell that has died from apoptosis, which provides information exceptionally useful in the clinical field, in particular in neurodegenerative, vascular and oncological diseases.